Methods of Treating Autoimmune Diseases

ABSTRACT

Novel methods for treating patients with autoimmune diseases are disclosed. The methods of the invention include first depleting circulating lymphocytes in the mammal, e.g., by administering anti-thymocyte antibody, and then, during the course of repopulation, administering to the mammal a therapeutically effective amount of latent TGF-β and/or another agent that promotes expansion of regulatory T cells. In certain aspects, the disclosed process results in improved kidney function and survival rates.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/680,667,filed Nov. 19, 2012, which is a continuation of U.S. application Ser.No. 12/241,345, filed Sep. 30, 2008, which is a continuation ofInternational Application PCT/US2007/066416, filed Apr. 11, 2007, whichclaims priority from U.S. Provisional Application 60/744,713, filed Apr.12, 2006, all of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

A paper copy of a sequence listing associated with this application isbeing submitted herewith and is hereby incorporated by reference in itsentirety into the specification.

FIELD OF THE INVENTION

This invention relates to methods of treating autoimmune diseases. Themethods of the invention involve the use of latent TGF-β or other agentsthat stimulate regulatory T cells, alone or in combination withlymphocyte-depleting agents, such as, e.g., anti-thymocyte globulin(ATG).

BACKGROUND OF THE INVENTION

The production of antibodies against self-antigens and/or autoreactive Tcells is a hallmark of many autoimmune diseases. Autoantibodies andautoreactive T cells can cause severe tissue damage (e.g., as in lupusnephritis) or loss of blood components (e.g., as in immunethrombocytopenia purpura).

Typically, autoimmune diseases are treated with nonspecificimmunosuppressive agents, such as, e.g., cyclophosphamide, methotrexate,azathioprine, and cyclosporine, that impede the immune cells fromattacking the organs and tissues. However, immunosuppressive agents areoften associated with significant side effects (e.g., toxicity, theundesired suppression of the immune system, etc.).

Due to its immunosuppressive effects, transforming growth factor-beta(TGF-β) has been suggested as a possible therapeutic agent for certainautoimmune diseases, including multiple sclerosis and graft-versus-hostdisease. Flanders et al., Clin. Med. Res., 1:13-20 (2003). It has alsobeen reported as useful to induce the generation of suppressor T cellsin vitro (see, e.g., U.S. Pat. No. 6,759,035). However, TGF-β is apluripotent cytokine—besides having immunosuppressive properties, it isinvolved in the extracellular matrix production, and other biologicalprocesses. For a review on TGF-β, see, e.g., Cytokine Reference, eds.Oppenheim et al., Academic Press, San Diego, Calif., 2001. Excessive orpersistent expression of TGF-β plays a role in organ fibrosis (Kapanciet al., Am. J. Resp. Crit. Care Med., 152:2163-2169 (1995); George etal., Prot. Natl. Acad. Sci., 96:2719-12724 (1999); Kuwahara et al.,Circulation, 106:130-135 (2002)), while systemic administration ofactive TGF-β has been associated with unacceptable toxicity. Inparticular, in a Phase I/II clinical trial for chronic progressivemultiple sclerosis, systemic administration of active TGF-β2 resulted inunacceptable renal toxicity as evidenced by a reduction in glomerularfiltration rate. Calabresi et al., Neurology, 51:289-292 (1998). Thisresult has hindered further clinical development of therapies involvingsystemic administration of active TGF-β. Accordingly, the challenge ofselectively harnessing the immunosuppressive potential of TGF-β withoutincurring its attendant toxicities has remained. In addition, thereremains a need to develop methods of treating autoimmune diseases thatallow suppression of autoreactive immunity without undesirable sideeffects.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery anddemonstration that latent TGF-β may be used to circumvent systemictoxicity of active TGF-β. Activation of latent TGF-β requires removal ofthe latency-associated peptide (LAP) which can occur in vivo through anumber of mechanisms including proteolytic cleavage, exposure toreactive oxygen species, and interactions with thrombospondin and otherproteins. Murphy-Ullrich et al., Cytokine Growth Factor Rev., 11:59-69(2000). It is theorized, but not relied on for the purposes of thisinvention, that such conditions are likely to occur in areas ofautoimmune inflammation, such as in the kidney in lupus patients.Because the activation of latent TGF-β occurs in areas of inflammationand tissue injury, the use of latent TGF-β may avoid the toxicityassociated with systemic TGF-β. Accordingly, in some embodiments, themethods of the invention involve systemic administration of inactiveTGF-β (e.g., latent TGF-β) to a mammal, whereupon the activation and/oraction of TGF-β is limited to sites of inflammation and tissue damage.

The present invention is further based, in part, on the discovery anddemonstration that depletion of lymphocytes by anti-thymocyte globulin(ATG) followed by administration of latent TGF-β is effective inimproving kidney function and increasing survival rates in a murinemodel of systemic lupus erythematosus. Accordingly, in some embodimentsof the invention, host lymphocytes are depleted prior to theadministration of latent TGF-β so as to yield the therapeuticallydesired effect of the latent TGF-β administration.

It is further theorized, but not relied on for the purposes of thisinvention, that the therapeutic effect of TGF-β is achieved, in part,due to the stimulatory effects of TGF-β on the growth of regulatory Tcells. Therefore, in some embodiments of the invention, another agentthat promotes the expansion of regulatory T cells may be administered inplace of, or in addition to, latent TGF-β.

This invention provides methods for treating a mammal (e.g., a human)with an autoimmune disease, e.g., systemic lupus erythematosus (SLE),rheumatoid arthritis (RA). In some embodiments, the treatment results inslowing the progression of disease and/or improvement in symptoms. Theinvention further provides methods of preserving or improving kidneyfunction in a mammal with an autoimmune disease that impairs kidneyfunction, such as, e.g., SLE, Goodpasture's syndrome, Wegener'ssyndrome, and Berger's disease.

In more particular embodiments, the methods of the invention include thefollowing steps:

(a) depleting circulating lymphocytes in a mammal,

(b) allowing the lymphocytes to begin repopulating (“repopulationphase”), and

(c) during the repopulation phase, administering to the mammal atherapeutically effective amount of latent TGF-β and/or an agent thatpromotes the expansion of regulatory T cells.

In some embodiments, the depletion of lymphocytes is accomplished byadministering anti-thymocyte antibody (e.g., Thymoglobulin®, Atgam™,Fresenius™, and Tecelac™) or another antibody specific for an antigen(s)expressed on T cells.

Once the circulating lymphocytes have been substantially depleted, theyare allowed to start repopulating (“repopulation phase”). During thecourse of repopulation, before the complete repopulation occurs, atherapeutically effective amount of one or more of the following agentsis administered to the mammal: (1) latent TGF-β (e.g., the latent formof any one of TGFβ1-TGFβ3) and/or (2) one or more other agents thatpromotes expansion of regulatory T cells (e.g., IL-10, IL-10 and IL-4,IL-10 and IFN-α, vitamin D3 and dexamethasone, vitamin D3 andmycophenolate mofetil, and rapamycin).

In some embodiments, where the kidney function is compromised due toautoimmune disease, the treatment methods result in improvement ofkidney function in the mammal (e.g., slowing the loss thereof) asindicated by, e.g., a change in systemic blood pressure, proteinuria,album inuria, glomerular filtration rate, and/or renal blood flow.

The foregoing summary and the following detailed description areexemplary and explanatory only and are not restrictive of the invention,as claimed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of amino acid sequences of the precursors ofhuman TGF-β1 (SEQ ID NO:1), TGF-β2 (SEQ ID NO:2), and TGF-β3 (SEQ IDNO:3). TGF-β2 is shown in the ‘long’ alternatively spliced form in whicha 28 amino acid insertion is found in the pre-pro domain beginning atresidue 119. Conserved sequences are boxed in. Arrows indicate the sitesof proteolytic processing resulting in cleavage of the signal peptideand of the mature C-terminal TGF-β1 fragment. * refers to RGD integrinrecognition site found in the latency-associated peptide (LAP) proteinsof TGF-β1 and TGF-β3. + refers to cysteine residues involved indisulfide bonds between the two monomeric LAP proteins. # refers to acysteine residue involved in formation of the single disulfide bondTGF-β monomers.

FIG. 2 shows the therapeutic effect of the ATG/latent TGF-β1 combinationtreatment on kidney function. MRUMPJ-Tnfrs6^(lpr) mice (a murine modelof SLE) were injected with 500 μg of ATG intraperitoneally (i.p.) twice,three days apart, with or without 4 μg of latent TGF-β1 in 100 μl ofphosphate buffered saline (PBS). Four micrograms of latent TGF-β1corresponds to a 1 μg (˜0.05 mg/kg) dose of the active (mature,non-LAP-associated) portion of the molecule. When included in atreatment, the latent TGF-β1 was administered daily for twelve daysbeginning eleven days after the second ATG injection. As a negativecontrol, SLE mice were treated with 500 μg of normal rabbitimmunoglobulin (Ig) i.p. twice, three days apart. An additionaltreatment group received normal rabbit immunoglobulin and latent TGF-β1administered as above. As a positive control, SLE mice were treated with100 mg/kg i.p. of cyclophosphamide in 200 μl saline weekly. Proteinuriawas significantly lower in SLE mice treated with latent TGF-β1 and ATGas compared to SLE mice treated with either ATG alone, controlIg+TGF-β1, or control Ig alone. Mean total urine protein of thecombination treatment group approached the level achieved withcyclophosphamide, a current treatment for lupus.

FIG. 3 shows the effect of the combination treatment on the developmentof severe kidney disease. Mice were treated as described above for FIG.2. SLE mice treated with ATG and latent TGF-β1 together exhibited adecrease in the incidence of severe proteinuria (>500 mg/dl/day) ascompared to SLE mice treated with either ATG alone, control Ig+TGF-β1,or control Ig alone.

FIG. 4 shows the effect of the combination treatment on kidney function.Mice were treated as described above for FIG. 2. The mean urine albuminlevels were decreased in SLE mice treated with the combination of ATGand latent TGF-β1 in comparison with SLE mice treated with either ATGalone, control Ig+TGF-β1, or control Ig alone. ATG and latent TGF-β1combination treatment resulted in mean urine albumin levels near thoseachieved with cyclophosphamide treatment.

FIG. 5 shows the effect of the combination treatment on the developmentof severe kidney disease. Mice were treated as described above for FIG.2. The percent of SLE mice having severe albuminuria (>10 mg/dl/day) wasdecreased in the combination treatment group in comparison with eitherATG alone, control Ig+TGF-β1, or control Ig alone.

FIGS. 6A-6E show the effect of the combination treatment on thedevelopment of autoantibodies. Arrows indicate start of treatment. Micewere treated as described above for FIG. 2 and indicated accordingly inFIGS. 6A-6E. Overall, SLE mice treated with ATG and latent TGF-β1 showeda considerable delay in the rise of IgG anti-dsDNA antibody titers incomparison to mice treated with either ATG alone, control Ig+TGF-β1, orcontrol Ig alone.

FIG. 7 shows the effect of the combination treatment on survival of SLEmice. Mice were treated as described above for FIG. 2. SLE mice treatedwith ATG and latent TGF-β1 survived significantly longer than SLE micetreated with either ATG alone, control Ig+TGF-β1, or control Ig alone.

FIG. 8 shows the survival data obtained in a repeat study withMRUMPJ-Tnfrs6^(lpr) mice treated as described above for FIG. 2. In thisinstance, the study was extended to 40 weeks of age (as opposed to 24weeks in the first study) to assess the durability of the effect oftransient treatment with ATG and latent TGF-β1. The survival benefit didin fact persist and the survival of mice treated with ATG and latentTGF-β1 was comparable to that obtained with cyclophosphamide, thepositive control (90% vs. 100%, respectively).

FIG. 9A shows the absolute number of CD4⁺ CD25⁺ cells in cultures ofsplenocytes exposed to various treatments. Splenocytes were pooled fromten MRL/lpr mice with active disease. Six different conditions (8wells/condition) were assayed: 1) cells alone, 2) ATG (100 μg/ml)+activeTGF-β1 (10 ng/ml; Genzyme), 3) ATG alone (100 μg/ml), 4) control rabbitIgG (100 μg/ml)+active TGF-β1 (10 ng/ml), 5) control rabbit IgG alone(100 μg/ml), and 6) active TGF-β1 alone (10 ng/ml). After five days, thereplicates of each culture condition were pooled, washed in phosphatebuffered saline, counted, and stained for FACS analysis. A sample of5×10⁵ cells per treatment was stained with rat anti-mouse CD4-Alexa 488and rat anti-mouse CD25-PerCp-Cy5.5 and analyzed by flow cytometry. Anacquisition of 6,000 lymphocytes per treatment was analyzed for stainingon a FACS Calibur system (Becton Dickinson, San Diego, Calif.). Resultsare expressed as the absolute number of cells of each phenotyperecovered under each culture condition (percent positive cells byFACS×total number of cells recovered from the culture).

FIG. 9B shows the absolute number of CD4⁺ CD25⁺ FOXP3⁺ cells in culturesof splenocytes treated as described for FIG. 9A. Additionally, forintracellular detection of FOXP3, cells stained for surface CD4/CD25were permeabilized overnight and stained for FOXP3.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods of treating a mammal with an autoimmunedisease. In particular embodiments, such methods include methods ofimproving kidney function in a mammal with an autoimmune disease thatcompromises kidney function. In some embodiments, the methods of theinvention involve systemic administration of latent TGF-β to a mammal,wherein the activation and/or action of TGF-β is limited to sites ofinflammation and tissue damage.

In some embodiments, methods of the invention comprise the followingsteps:

(a) depleting circulating lymphocytes in the mammal,

(b) allowing the lymphocytes to begin repopulating, and

(c) during the repopulation phase of (b), administering to the mammal atherapeutically effective amount of latent TGF-β and/or an agent thatpromotes the expansion of regulatory T cells.

Lymphocyte Depletion

Depletion of circulating lymphocytes can be accomplished byadministering a lymphocyte-depleting agent to the mammal or otherwiseexposing the mammal to conditions that result in a loss of a substantialfraction of lymphoid cells (e.g., lymphocytes, natural killer (NK)cells, monocytes, and/or dendritic cells, etc.) in the mammal.Lymphocytes to be depleted may be T lymphocytes (T cells) and/or T and Blymphocytes. In the depletion phase, T cell counts are reduced by atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, and optionally, Blymphocyte (B cell) counts are reduced by at least 30%, 40, 50%, 60%,70%, 80%, 90%, 95%, or more. In preferred embodiments, the depletedlymphocytes are predominantly T cells, which means that the percentageof depleted T cells is greater (e.g., 1.2-, 1.5-, 2-, 5-, 10-fold, ormore) than the percentage of depleted B cells.

The level of lymphocyte depletion can be readily assessed by, forexample, measuring the amount of peripheral blood lymphocytes (PBLs).Lymphocyte counts can be determined using conventional clinicallaboratory techniques (e.g., by flow cytometry). Reference values fornormal PBL levels in humans are presented in Table 1.

TABLE 1 Typical Mean Range Mean Range Cell Type Marker (%) (%)(cells/μl) (cells/μl) Total T cells CD3 71 55-87 1,586 781-2,391 Total Bcells CD19 5 1-9 277 17-537  Helper T cells CD4 43 24-62 1,098 447-1,750Cytotoxic cells CD8 42 19-65 836 413-1,260

In some embodiments, the lymphocyte-depleting agent is ananti-lymphocyte antibody, e.g., anti-T cell antibodies, e.g.,anti-thymocyte globulin (ATG), such as, e.g., Thymoglobulin®, Atgam™Fresenius™, and Tecelac™ ATG is a polyclonal antibody directed againstthymocytes. Currently marketed ATG products are produced by injectingthymocytes from one species (e.g., human) into another species (e.g.,rabbit or horse). ATG binds to cell surface proteins such as lymphocytesurface antigens CD2, CD3, CD4, CD8, CD11a, CD18, CD25, HLA DR, and HLAclass I (Bourdage et al., Transplantation, 59:1194-1200 (1995)). ATG isbelieved to induce immunosuppression primarily as a result of T celldepletion (see, e.g., Bonnefoy-Bernard et al., Transplantation,51:669-673 (1991)) and has been previously used for pretreatingtransplant patients to reduce the risk of rejection in the context oforgan transplantation.

In addition to ATG, the lymphocyte-depleting agent consists of orcomprises a monoclonal or polyclonal antibody directed to one or morespecific lymphocyte surface antigens, e.g., anti-CD52 antibody (e.g.,Campath®), anti-CD3 antibody (e.g., OKT3®), anti-CD4 antibody (OKT™),anti-CD25 (IL-2R) antibody (e.g., daclizumab), anti-CD5 antibody,anti-CD7 antibody, anti-TCR antibody, anti-CD2 (e.g., Siplizumab™), oran antibody against any of other lymphocyte surface antigens specifiedabove, etc.

In some embodiments, the lymphocyte-depleting agent is a corticosteroid.

In some embodiments, conditions that result in depletion of lymphocytesinclude exposure to gamma radiation.

A combination of any suitable agents and/or conditions to depletelymphocytes can be also used.

Reconstitution Phase

Following the depletion phase, the lymphocytes of the mammal are allowedto begin repopulating by withdrawing the lymphocyte-depleting agent ormitigating the conditions that resulted in the loss of lymphocytes.

While in some instances, the agent of step (c) (i.e., TGF-β or anotheragent that specifically stimulates regulatory T cells) can beadministered to the mammal immediately at the start of the replenishmentphase, in other cases, the agent is administered after some repopulationhas occurred. Before the step (c) agent is administered to the mammal,the lymphocytes may be allowed to repopulate to less than 50%, 40%, 30%,20%, 10%, 5%, or lower, as compared to the pre-depletion level.

In humans, lymphocytes repopulate to pre-depletion levels at differentrates depending on the depleting agent. For example, with ATG, acomplete repopulation may take two to four months, while after treatmentwith Campath™, the repopulation may take several years. Accordingly, insome embodiments, the length of time between the end of the depletionphase of the lymphocytes and the administration of step (c) agent is,for example, 0, 1, 2, 3, 4, 5, 6 days; 1, 2, 3, 4, or 5 weeks, orlonger.

TGF-β

In certain embodiments, the methods of the invention involveadministration of inactive TGF-β which is activated afteradministration. In some embodiments, inactive TGF-β is administered inthe form of latent TGF-β. In other embodiments, inactive TGF-β isadministered in the form of a TGF-β-encoding DNA which expresses activeTGF-β upon induction.

TGF-β is naturally secreted in either a so-called “small latent complex”(100 kDa) in which the biologically active TGF-β is noncovalentlyassociated with its pro domain (“latency-associated peptide,” LAP) andin a so-called “large latent complex” (220 kDa) additionally containinglatent TGF-β biding protein (LTBP). The latent forms are unable to bindto TGF-β receptors until active, i.e., mature, TGF-β is released fromthe complex. For a more detailed review of the latent forms andactivation process, see, e.g., Cytokine Reference, eds. Oppenheim etal., Academic Press, San Diego, Calif., 2001, pp. 724-725. As usedherein, the term “latent TGF-β” refers to TGF-β associated with LAP(covalently or noncovalently) and, optionally, additionally associatedwith LTBP (covalently or noncovalently). The term, therefore, refers tosmall and large latent TGF-β complexes. Other forms of inactive TGF-βthat could be activated in the locations and at the time periods desiredwould also be useful in the methods of this invention. There are threeknown mammalian isoforms of TGF-β (TGF-β1 to TGF-β3), all of which arehomologous among each other (60-80% identity). A partial listing ofprotein accession number for the three mammalian isoforms is provided inTable 2; an alignment of human TGF-βs is shown in FIG. 1.

TABLE 2 Species TGF-β1 TGF-β2 TGF-β3 Human PO1137 PO8112 P109600 MouseP04202 P27090 P171125 Rat AAD20222 AAD24484 Q07258 Porcine AAA616AAB03850 P15203 Simian P09533 WFMKB2

The structural and functional aspects of TGF-β as well as TGF-βreceptors are well known. See, e.g., Cytokine Reference, eds. Oppenheimet al., Academic Press, San Diego, Calif., 2001. Thus, inactivated formsof engineered TGF-βs that retain the ability to bind to one or moreTGF-β receptors (TGF-βRI, TGF-βRII, or TGF-βRIII) would also be usefulin the methods of the invention. Such inactivated forms of engineeredTGF-β may contain only a partial or a mutated amino acid sequence of thenaturally occurring TGF-β. For example, inactivated forms of engineeredTGF-β may contain native sequences in which conservative substitutionswere made and/or nonessential amino acids were deleted. For example,they may comprise a sequence, which is at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to the 112 amino acid C-terminalportion of SEQ ID NO:n over the entire length of this C-terminal portionof SEQ ID NO:n, wherein n=1, 2, or 3.

Agents that Promote Regulatory T Cell Expansion

In certain embodiments, the methods of the invention involveadministration of an agent that promotes regulatory T cells expansion.Regulatory T cells (also known as Tregs or suppressor T cells) are cellsthat are capable of inhibiting the proliferation and/or function ofother lymphoid cells via contact-dependent or contact-independent (e.g.cytokine production) mechanisms. Several types of regulatory T cellshave been described, including γδ T cells, Natural Killer T (NKT) cells,CD8⁺ T cells, CD4⁺ T cells, and double negative CD4⁻ CD8⁻ T cells. See,e.g., Bach et al., Immunol., 3:189-98 (2003). The so-called “naturallyoccurring” regulatory T cells are CD4⁺ CD25⁺ and express the forkheadfamily transcription factor FOXP3 (forkhead box p3). In addition to theFOXP3-expressing CD4⁺ CD25⁺, a minor population of CD8⁺ FOXP3-expressingcells are also regulatory T cells. CD4⁺ Tregs can be further dividedinto induced regulatory T cells that secrete interleukin-10 (IL-10) andTGF-β such as Tr1 cells and T-helper 3 (Th3) cells. Additional surfacemarkers for CD4⁺ CD25⁺ regulatory T cells include CD45RB, CD38, GITR,surface TGF-β, CTLA4, CD103, CD134 and CD62L. For a detailed review ofvarious types of regulatory T cells, see, e.g., Wing et al., Scand. J.Immunol., 62(1):1 (2005); Jonuleit et al., J. Immunol., 171:6323-6327(2003); Horwitz et al., J. Leukocyte Biol., 74:471-478 (2003).

Accordingly, in some embodiments, the regulatory T cells that are beingstimulated include one or more of the following groups: (1) regulatory Tcells that express IL-10; (2) regulatory T cells that express TGF-β(including Tr1 cells and Th3 cells); (3) CD4⁺ CD25⁺ cells (includingcells having additional markers CD45RB⁺, CD38⁺, GITR, surface TGF-β,CTLA-4, CD103, CD134 and/or CD62L); (4) FOXP3-expressing T cells(including CD8⁺ cells and CD4⁺ cells); (5) γδ T cells; (6) NK T cells;and (7) double negative CD4⁻ CD8⁻ T cells.

TGF-β, in addition to its direct immunosuppressive activity, may also becapable of stimulating regulatory T cells. Gorelik and Flavell, NatureReviews Immunology, 2:46-53 (2002); Chen et al., J. Exp. Med.,198:1875-1886 (2003); Marie et al., J. Exp. Med., 7:1061-1067 (2005);Huber et al., J. Immunol., 173:6526-6531 (2004).

Examples of agents, other than TGF-β, that promote regulatory T cellexpansion include: (1) IL-10; (2) IL-10 and IL-4; (3) IL-10 and IFN-α;(4) vitamin D3 and dexamethasone; (5) vitamin D3 and mycophenolatemofetil, and (6) rapamycin. (See, e.g., Barrat et al., J. Exp. Med.,195:603-616 (2002); Jonuleit et al., J. Immunol., 171:6323-6327 (2003);Gregori et al., J Immunol., 167:1945-1953 (2001); Battaglia et al.,Blood, 105:4743-4748 (2005).)

In some embodiments, an increase of, e.g., at least 10%, 20%, 30%, 40%,50%, 100%, or more in the expansion of regulatory T cells in thepresence of an agent as opposed to its absence is considered indicativeof the agent's capacity to promote regulatory T cells expansion. TGF-βand other agents can be assayed for their capacity to promote regulatoryT cell expansion using routine methods. Examples of some of the morefrequently used in vitro assays include the following:

(1) flow cytometry analysis, wherein co-expression of CD4, CD25, and/orFOXP3, and/or CD62L, and/or GITR, and/or CTLA4, and/or surface TGF-β,and/or CD103, and/or CD134 is used as indication of a regulatory T cellphenotype (see, e.g., Jonuleit, supra);

(2) inhibition of T cell proliferation in a co-culture system asdescribed in, e.g., Chen et al., J. Exp. Med., 198:1875-1886 (2003). (Inthis assay, regulatory T cells are added to responder T cells and theco-culture is stimulated with anti-CD3 or allogeneic lymphocytes. In thepresence of regulatory T cells, the responder T cells become unable toproliferate in response to these stimuli. The degree of proliferation istypically measured by tritiated thymidine incorporation.); and

(3) cytokine profiling as described in, e.g., Barrat, supra, andJonuleit, supra. (In this assay, a supernatant from cultured regulatoryT cells is analyzed for the presence of the immunosuppressive cytokinessuch as, e.g., IL-10 and TGF-β, known to be produced by regulatory Tcells.)

Uses

The methods of the invention can be used to treat a mammal that has anautoimmune disease such as, e.g., systemic lupus erythematosus (SLE) andautoimmune rheumatoid arthritis (RA). Examples of mammals include humansor other primates (e.g., chimpanzees), rodents (e.g., mice, rats, orguinea pigs), rabbits, cats, dogs, horses, cows, and pigs. In some ofthe subjects afflicted, the treatment is expected to result ininhibiting the progression of disease and/or improvement in symptoms.

Examples of additional autoimmune diseases include insulin-dependentdiabetes mellitus (IDDM; type I diabetes), inflammatory bowel disease(IBD), graft-versus-host disease (GVHD), celiac disease, autoimmunethyroid disease, Sjögren's syndrome, autoimmune gastritis, autoimmunehepatitis, cutaneous autoimmune diseases, autoimmune dilatedcardiomyopathy, multiple sclerosis (MS), myasthenia gravis (MG),vasculitis (e.g., Takayasu's arteritis and Wegener's granulomatosis),autoimmune diseases of the muscle, autoimmune diseases of the testis,autoimmune ovarian disease, autoimmune uveitis, Graves' disease,psoriasis, ankylosing spondylitis, Addison disease, Hashimotothyroiditis, idiopathic thrombocytopenic purpura, and vitiligo.

The methods of the invention are expected to slow the progression ofautoimmune disease, improve at least some symptoms, and/or increasesurvival. For example, the methods of the invention may result in areduction in the levels of autoantibodies, B cells producingautoantibodies, and/or autoreactive T cells. The reduction in any ofthese parameters can be, for example, at least 10%, 20%, 30%, 50%, 70%or more as compared to pretreatment levels.

The invention further provides methods of preserving or improving kidneyfunction in a mammal with an autoimmune disease that compromises kidneyfunction. Examples of autoimmune diseases that may compromise kidneyfunction include SLE (e.g., lupus nephritis), Goodpasture's syndrome,Wegener's granulomatosis (Wegener's syndrome), Berger's disease (IgAnephropathy), and IgM nephropathy. In some of the patients afflictedwith such diseases, the treatment is expected to result in improvementof kidney function (e.g., slowing the loss of, preserving, or improvingthe same) as indicated by, e.g., a change in systemic blood pressure,proteinuria, albuminuria, glomerular filtration rate, and/or renal bloodflow.

The term “renal function” refers to the ability of a kidney to performits physiological functions such as pressure filtration, selectivereabsorption, tubular secretion, and/or systemic blood pressureregulation. Methods for assessing renal function are well known in theart and include, but are not limited to, measurements of blood systemicand glomerular capillary pressure, proteinuria, albuminuria, microscopicand macroscopic hematuria, serum creatinine level (e.g., one formula forestimating renal function in humans equates a creatinine level of 2.0mg/dl to 50% of normal kidney function and 4.0 mg/dl to 25%), decline inthe glomerular filtration rate (GFR) (e.g., as indicated by the rate ofcreatinine clearance, or using inulin assays), and degree of tubulardamage.

For a detailed review of renal function and related disease states, seeThe Kidney: Physiology and Pathophysiology, eds. Seldin et al., 3^(rd)ed., Lippincott, Williams & Wilkins Publishers, 2000. Normally, lessthan 0.15 g of protein is excreted into the urine in a 24-hour period.Almost all types of kidney disease cause mild (up to 500 mg per day) tomoderate (up to 4 g per day) protein leakage into the urine. The normalconcentration of albumin in the urine is less than 1.0 mg/dl. Generally,30-300 mg/dl urinary albumin is considered microalbuminuria, and greaterthan 300 mg/dl is considered macroalbuminuria. The normal values ofserum creatinine are 0.6-1.5 mg/dl for men and 0.6-1.1 mg/dl for women.The relationship between creatinine levels, renal function, and thestage of renal disease is shown in Table 3.

TABLE 3 Estimated Creatinine Reduction of Level (mg/dl) Renal FunctionStage of Renal Disease 0.6-1.5 Up to 25% Reduced or diminished renalreserve >1.5 >50%  Renal insufficiency 4.8 75% Renal failure 10 90%End-stage renal disease

Therefore, the methods of the invention may be useful in patients havingan autoimmune disease with reduced or diminished renal reserve, renalinsufficiency, renal failure, or end-stage renal disease. For example,methods of the invention may be used in patient with microalbuminuria,macroalbuminuria, and/or proteinuria levels over 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 g or more per a 24-hour period, and/or serum creatinine levelsof about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0,8.0, 9.0, 10 mg/dl or higher.

In some embodiments, the methods of the invention reduce the amount ofprotein secreted in the urine (proteinuria), amount of albumin secretedin the urine (albuminuria), and/or the patient's serum creatinine levelsby at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more, relative tocontrol subjects. In other embodiments, the methods of the inventionslow the loss of renal function by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, or more, relative to control subjects. Nonlimitingillustrative methods for assessing renal function are described hereinand, for example, in WO 01/66140.

Methods of Administration

In the methods of the invention, “administration” is not limited to anyparticular delivery system and may include, without limitation,parenteral (including subcutaneous, intravenous, intramedullary,intraarticular, intramuscular, or intraperitoneal injection), rectal,topical, transdermal, or oral (for example, in capsules, suspensions, ortablets). Administration to an individual may occur in a single dose orin repeat administrations, and in any of a variety of physiologicallyacceptable salt forms, and/or with an acceptable pharmaceutical carrierand/or additive as part of a pharmaceutical composition. Physiologicallyacceptable salt forms and standard pharmaceutical formulation techniquesand excipients are well known to persons skilled in the art (see, e.g.,Physicians' Desk Reference (PDR®) 2005, 59^(th) ed., Medical EconomicsCompany, 2004; and Remington: The Science and Practice of Pharmacy, eds.Gennado et al. 21th ed., Lippincott, Williams & Wilkins, 2005).

Latent TGF-β can also be administered by means of gene therapy (i.e., byadministering a TGF-β-encoding DNA in an appropriate vector), forexample, as described in Kitani et al., J. Exp. Med., 192(1):41-52(2000).

The appropriate effective doses for the latent TGF-β, agents promotingTregs, and lymphocyte depleting agents will be chosen by a treatingclinician and will range roughly from 0.01 μg/kg to 25 mg/kg, from 0.1μg/kg to 10 mg/kg, from 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, from 10μg/kg to 100 μg/kg, from 100 μg/kg to 1 mg/kg, and from 500 μg/kg to 5mg/kg. Additionally, specific dosages indicated in the Examples or inthe PDR® 2005 and later editions may be used to arrive at the desireddosage. For example, the currently approved uses of Thymoglobulin® inthe United States include transplantation (from 1 mg/kg to 2.5 mg/kg for2-14 days) and aplastic anemia (from 2.5 mg/kg to 3.5 mg/kg for 5 days).

Effective dosages achieved in one animal may be converted for use inanother animal, including humans, using conversion factors known in theart. See, e.g., Freireich et al., Cancer Chemother. Reports,50(4):219-244 (1966) and Table 4 for equivalent surface area dosagefactors. Examples of autoimmune disease models and appropriate methodscan be found in, e.g., Cohen et al. (eds.) Autoimmune Disease Models,Academic Press, 2005.

TABLE 4 From: Mouse Rat Monkey Dog Human To: (20 g) (150 g) (3.5 kg) (8kg) (60 kg) Mouse 1 0.5 0.25 0.17 0.08 Rat 2 1 0.5 0.25 0.14 Monkey 4 21 0.6 0.33 Dog 6 4 1.7 1 0.5 Human 12 7 3 2 1

The following Examples are provided for illustrative purposes and arenot intended to be limiting.

EXAMPLES Potency Assessment of Activated TGF-β1

Recombinant human latent TGF-β1 was produced in CHO cells (Genzyme,Framingham, Mass.). Disruption of LAP from latent TGF-β1 was achievedthrough acidification. LAP-TGF-β1 was diluted to 200 ng/mL in assaymedium (DMEM plus non-essential amino acids, L-glutamine, pen-strep, and10% FBS). Five hundred microliters of the diluted sample was activatedby adding 100 μL of 1N HCl and incubating at room temperature for 20minutes. The sample was subsequently neutralized with 100 μL of 1.2 NNaOH/0.5 M HEPES.

The activated TGF-β1 sample was analyzed using the A549 Cell PotencyAssay and the activity assessed in comparison to a human recombinantTGF-β2 (Genzyme, Framingham, Mass.) control. The A549 potency assay isbased on the TGF-β1-induced release of IL-11 by the human lungepithelial cell line, A549 and is described in Wang et al., Am. J.Physiol., 276:L175-L185 (1999). IL-11 release from the A549 cells inresponse to TGF-β1 was measured using an ELISA procedure (R&D Systems,Minneapolis, Minn.).

Murine Lupus Model

Animals and Reagents—

Female MRL/lpr mice were obtained from the Jackson Laboratory (BarHarbor, Me.) and were received at 5-6 weeks of age. ATG was generated bythe immunization of rabbits with Balb/c mouse thymocytes as follows.Rabbits were immunized subcutaneoulsy with 5×10⁷ fresh thymocytes on day0 and boosted intravenously with 5×10⁷ fresh thymocytes on day 14. Serumcollected on days 20, 22, and 25 was pooled and the IgG fraction wasisolated by chromatography and sodium sulfate precipitation. Acommercial preparation of IgG from naïve rabbits was used as a negativecontrol (Sigma, St. Louis, Mo.). Recombinant human latent TGF-β1 wasproduced in CHO cells (Genzyme, Framingham, Mass.). Cyclophosphamide waspurchased from VWR Scientific Products (West Chester, Pa.).

Treatment—

Animals were monitored for proteinuria, albuminuria, and titers of IgGantibodies to double-stranded DNA (dsDNA) every three weeks (see below).Therapeutic treatment was initiated when animals started developingantibodies to dsDNA and/or elevated proteinuria at 12-13 weeks of age.Treatment with ATG or control rabbit IgG consisted of twointraperitoneal (i.p.) injections of 500 μg (˜25 mg/kg) delivered threedays apart (days 0 and 3). Latent TGF-β1 was given from days 14-25 astwelve daily i.p. injections of 4 μg per mouse. A 4 μg dose of latentTGF-β1 corresponds to a 1 μg (˜0.05 mg/kg) dose of the active (mature,non-LAP-associated) portion of the molecule. Cyclophosphamide was usedas a positive control and was delivered i.p. weekly at a dose of 100mg/kg from 12-13 weeks of age until the end of the study at 24-25 weeksof age. The treatment groups consisted of control rabbit IgG, controlrabbit IgG+latent TGF-β1, ATG, ATG+latent TGF-β1, or cyclophosphamidewith ten animals per group.

Proteinuria and Albuminuria—

Levels of protein in the urine of individual mice were measured using acolorimetric assay designed to measure total protein concentration.Levels of albumin in the urine were assessed with a quantitative ELISAassay.

A 24-hour urine collection was performed every three weeks by placingmice into individual metabolic cages. Proteinuria was measured using theMicroprotein-PR™ kit from Sigma (St. Louis, Mo.) according tomanufacturer's instructions. Briefly, urine was added to a reagentsolution containing pyrogallol red-molybdate complex. The mixture wasincubated at 37° C. for ten minutes to allow for binding of the reagentto basic amino groups on proteins leading to a shift in absorbance at600 nM. The increase in optical density (O.D.) at 600 nM is directlyproportional to protein concentration and a reference standard was usedto calculate the protein concentration of test samples according to thefollowing formula:

${\frac{{OD}_{sample}}{{OD}_{standard}} \times {Conc}_{standard} \times {Dilution}} = {{Conc}_{sample}\left( {{mg}\text{/}{dl}} \right)}$

Levels of albumin in the urine were assessed using an indirectcompetitive ELISA kit, according to manufacturer's instructions(Albuwell M from Exocell Inc, Philadelphia, Pa.). Briefly, serialtwo-fold dilutions of urine samples were added to duplicate wells of anELISA plate coated with mouse albumin. Rabbit anti-mouse albuminantibody was then added to the wells allowing for competition betweenbinding of the antibody to albumin in the sample and albumin attached tothe well. This was followed by the addition of horseradish peroxidase(HRP)-conjugated anti-rabbit immunoglobulin and HRP substrate to detectthe amount of rabbit anti-mouse albumin antibody bound to the well. TheO.D. at 450 nm was inversely proportional to the logarithm of the amountof albumin in the urine sample. The albumin concentration in the urinesamples was derived from a standard curve obtained with knownconcentrations of murine albumin.

Anti-dsDNA ELISA—

Titers of IgG antibodies to dsDNA in serum samples from individual micewere measured by ELISA.

Serum samples from individual mice were collected every three weeks.Titers of antibodies to dsDNA were assessed by ELISA. Mousedouble-stranded DNA (The Jackson Laboratory) was digested with S1nuclease (Invitrogen, Carlsbad, Calif.) to remove any single-strandedDNA and was then used to coat the wells of a 96-well ELISA plate (100μl/well of 1 μg/ml dsDNA) overnight at 4° C. The plates were pretreatedwith 0.01% protamine sulfate in water (150 μl/well for 90 minutes atroom temperature) to facilitate adhesion of the DNA. After coating, theplates were incubated with 2.5% BSA blocking buffer for one hour at 37°C. and washed. One hundred microliters of serial two-fold dilutions ofserum were then added to duplicate wells and incubated at 37° C. for onehour. The plates were washed and HRP-conjugated goat anti-mouse IgG(Pierce, Rockford, Ill.) was added to detect antibodies bound to dsDNA(37° C. for one hour). After washing, HRP substrate was added for 30minutes at room temperature and the O.D. of the colorimetric product wasread at 490 nM with a reference wavelength of 650 nM on a dualwavelength plate reader (Molecular Devices, Sunnyvale, Calif.). Theantibody titer was defined as the reciprocal of the dilution of serumgiving an O.D. greater or equal to 0.1. Normal mouse serum was used as anegative control (titer <200, the lowest dilution tested) and serum fromaged MRL/lpr lupus mice was used as a positive control (titer of6400-25600).

Histology—

Kidneys were collected for histological analysis at the time ofscheduled sacrifice or during the course of the study from moribundanimals that required euthanasia. The kidneys were sliced longitudinallyand were fixed in neutral buffered formalin. Sections of approximately 5μm were stained with hematoxylin and eosin (H&E) and periodicacid-Schiff (PAS) stains. The slides were scored by pathologist forglomerular morphology, interstitial inflammation, and protein castsaccording to the scoring systems described in Tables 5A and 5B.

TABLE 5A Glomeruli 0 No significant lesions (comparable to WHO class I)1 Minimal to mild disease, characterized by mesangial deposits(comparable to WHO class IIA) 2 Mild to moderate disease, characterizedby hypercellularity with or without mesangial deposits (comparable toWHO class IIB) 3 Moderate to severe disease, characterized bymesangioproliferative glomerulopathy and “wire loop” capillaries with orwithout fibrinoid necrosis of capillary loops, rupture of Bowman'scapsule, and periglomerular inflammation and fibrosis (“crescent”formation) affecting less than 25% of the glomeruli. Focal synechiationof glomerular tuft to the Bowman's capsular epithelium is often presentand may be the only prominent finding; if synechiation is the onlyfinding, a score of 3 will be assigned if less than 75% of theglomerular tufts are affected. 4 Moderate to severe disease with samecharacteristics as score 3, but affecting 25-50% of the glomerular tufts5 Severe disease with same characteristics as score 3, but affecting50-75% of the glomerular tufts 6 Severe disease with samecharacteristics as score 3, but affecting greater than 75% of theglomerular tufts Scores 3-6 divide WHO scores III and IV into foursub-scores

TABLE 5B Interstitial Inflammation 0 No significant lesions 1 Minimal tomild inflammation and fibrosis 2 Mild to moderate inflammation andfibrosis 3 Moderate to severe inflammation and fibrosis 4 Severe anddiffuse inflammation and fibrosis Scores 0-4, based on density ofchronic inflammation (lymphocytes, plasma cells, and macrophages) withfibrosis within the interstitium and surrounding renal blood vessels

Statistics—

Statistical analysis was conducted using Tukey's multiple comparisontests to determine whether significant differences existed betweentreatment groups. P values equal to or less than 0.05 were accepted asstatistically significant.

Proteinuria and Albuminuria—

Treatment with ATG or latent TGF-β1 alone (control Ig+TGFβ1) largelyfailed to inhibit the development of proteinuria (FIG. 2), although bythe end of the study the incidence of severe proteinuria (>500mg/dl/day) was slightly reduced in these single agent treatment groupscompared to the mice treated with control Ig (60-67% vs. 90%,respectively) (FIG. 3). In contrast, treatment with a combination of ATGand latent TGF-β1 resulted in marked inhibition in the incidence (30%vs. 90%) and severity of proteinuria, suggesting a synergistic effectbetween these two agents (FIGS. 2 and 3).

The observed reduction in the levels of total protein in the urine ofmice treated with the combination of ATG and latent TGF-β1 was alsoreflected in the measurements of urine albumin levels (FIGS. 4 and 5).ELISA quantitation indicated that the incidence and severity of albuminuria was considerably reduced in mice treated with the combinedtherapy as compared either the negative control Ig group or the singleagent therapy groups (ATG, control Ig+TGFβ1).

Antibodies to dsDNA—

The majority of mice in the negative control group (normal rabbit Ig) aswell as the latent TGF-β1+control Ig and ATG-treated groups, graduallydeveloped rising titers of IgG antibodies against dsDNA with comparablekinetics. By comparison, there was a considerable delay in the rise ofanti-dsDNA titers in the group treated with the combination of ATG andlatent TGF-β1 (FIG. 6). Deposition of the immune complexes (DNA-anti-DNAcomplexes) in the glomeruli is believed to play an important role in theinflammation and renal pathology characteristic of lupus. However, theapparent inhibition in the development of antibodies to dsDNA in thecombination treatment group could not entirely account for thepreservation of kidney function, as there was a poor correlation betweentiters of antibodies and degree of proteinuria at the end of the study.

Survival—

Dosing with ATG and/or latent TGF-β1 was well tolerated and did not giverise to any obvious adverse events. All of the deaths, except for oneanimal in the latent TGF-β1+control Ig group, were associated with veryhigh levels of proteinuria and presumably resulted from kidney failure.All of the treatment groups showed an overall improvement in survivalwhen compared to the negative control rabbit Ig-treated group (FIG. 7).The highest degree of survival (100%) was seen in the cyclophosphamideand the ATG/latent TGF-β1 combination treatment groups, followed by theATG (90%) and latent TGF-β1+control Ig (70%) treatment groups. Thenegative control Ig group had only a 40% survival rate by the end of thestudy.

Histology—

The results of the histological analyses are presented in Table 6. Micetreated with ATG and latent TGF-β1 exhibited lesser degrees ofglomerulopathy compared to control mice or mice that received either ATGalone or latent TGF-β1 and control Ig. These histologic findingscorrelated with clinical findings of decreased proteinuria/albuminuriaand improved survival in the combination-treated animals.

A minimal decrease in inflammation scores was noted in groups treatedwith ATG, latent TGF-β1+control Ig and the combination of ATG and latentTGF-β1 in comparison to the group treated with control rabbit IgG alone.

TABLE 6 Treatment Group Glomeruli Score Inflammation Score ControlRabbit IgG 4.3 ± 1.4 2.8 ± 0.4 Latent TGF-β1 3.1 ± 0.8 2.6 ± 0.5 ATG 2.7± 0.5 2.4 ± 0.5 ATG + Latent TGF-β1 2.3 ± 0.7 2.7 ± 0.5 Cyclophosphamide1.0 ± 0.7 1.3 ± 0.5

A repeat study was performed with MRUMPJ-Tnfrs6^(lpr) mice following thesame treatment regimen as described above. In this instance, the studywas extended to 40 weeks of age (as opposed to 24 weeks in the firststudy) to assess the durability of the effect of transient treatmentwith ATG+latent TGF-β1. The results showed a long-term survival benefit.A 90% survival rate was observed in mice treated with ATG and latentTGF-β1 as compared to 30% survival in mice receiving control rabbit Ig,and 10% survival in the group treated with ATG. This compares favorablywith cyclophosphamide which provided 100% survival but required chronicweekly injections as opposed to a one time transient course of treatmentwith ATG+latent TGF-β1 (FIG. 8).

A similar study was conducted in NZB/NZWF1 mice, another model ofspontaneous lupus. The same treatment regimen was used and, under theseconditions, there was no statistically significant effect of treatmentwith ATG and latent TGF-β1 or either agent alone, on the course orseverity of disease. Due to differences in the characteristics andkinetics of disease between the two models, it is likely that thetreatment regimen needs to be optimized for the NZB/NZWF1 strain.

To investigate the mechanism of action underlying the activity ofATG+TGF-β1, spleen cells from MRL/lpr lupus mice were cultured in vitrowith ATG+/−TGF-β1 and the cells recovered were analyzed by FACS for thepresence of Tregs. Pooled spleen cells from ten MRL/lpr mice with activedisease (˜25 weeks old) were resuspended at 2×10⁶ cells/ml in serum-freeAIM-V medium (Gibco, Grand Island, N.Y.) supplemented with 100 U/mlpenicillin, 100 μg/ml streptomycin and 2 mM glutamine. The cells werecultured in 24-well plates containing 2 ml cells/well under sixdifferent conditions (8 wells/condition): 1) cells alone, 2) ATG (100μg/ml)+active TGF-β1 (10 ng/ml; Genzyme), 3) ATG alone (100 μg/ml), 4)control rabbit IgG (100 μg/ml)+active TGF-β1 (10 ng/ml), 5) controlrabbit IgG alone (100 μg/ml), and 6) active TGF-β1 alone (10 ng/ml).Active TGF-β1 was used to mimic the activation process that wouldnormally occur in vivo. The cells were incubated for five days at 37° C.and 5% CO₂. Cells from each culture condition were then pooled, washedin phosphate buffered saline, counted, and stained for FACS analysis. Atotal of 5×10⁵ cells per sample were stained with rat anti-mouseCD4-Alexa 488 (Cat. No. 557667; BD Pharmingen, San Diego, Calif.) andrat anti-mouse CD25-PerCp-Cy5.5 (Cat. No. 551071; BD Pharmingen). Forintracellular detection of FOXP3, cells stained for surface CD4/CD25were permeabilized overnight and stained using the eBioscience (SanDiego, Calif.) FOXP3 staining kit (Cat. No. 72-5775) according tomanufacturer's instructions. An acquisition of 6,000 lymphocytes pertreatment was analyzed for staining on a FACS Calibur system (BectonDickinson, San Diego, Calif.). Results are expressed as the absolutenumber of cells of each phenotype recovered under each culture condition(percent positive cells by FACS×total number of cells recovered from theculture).

As shown in FIG. 9, the number of CD4⁺ CD25⁺ T cells recovered was thegreatest in cultures containing ATG+TGF-β1. Regulatory T cells typicallyexpress a CD4⁺ CD25⁺ phenotype but activated T cells can also exhibitthis phenotype. Additional FOXP3 staining provides further evidence of aTreg phenotype and the results obtained confirmed that treatment withATG+TGF-β1 produced the greatest number of CD4⁺ CD25⁺ FOXP3⁺ Tregs.Treatment with ATG alone also appeared to lead to a slight increase inthis population (compared to cells alone) which was enhanced by theaddition of TGF-β1. These results support the hypothesis that treatmentwith ATG+TGF-β1 can promote the expansion of Tregs and that such cellsmay provide a therapeutic benefit under conditions of autoimmunity.

Murine Model of Arthritis

The effect of ATG+/−TGF-β1 was tested in a collagen-induced arthritismouse model. To induce disease, DBA/1 mice (Jackson Laboratory) wereimmunized on day 0 with bovine type II collagen (Cat. No. 2002-2,Chondrex) in complete Freund's adjuvant in a 100 μl total volume at thebase of the tail. A booster immunization with collagen in incompleteFreund's adjuvant was given on day 22. Treatment with ATG or controlrabbit IgG consisted of two intraperitoneal (i.p.) injections of 500 μg(˜25 mg/kg) delivered three days apart (days 23 and 26). Latent TGF-β1was given from days 28-37 as ten daily i.p. injections of 4 μg permouse. A 4 μg dose of latent TGF-β1 corresponds to a 1 μg (˜0.05 mg/kg)dose of the active (mature, non-LAP-associated) portion of the molecule.The treatment groups included (1) control rabbit IgG, (2) control rabbitIgG+latent TGF-β1, (3) ATG, and (4) ATG+latent TGF-β1, with ten animalsper group. Starting on day 21, individual mice were examined and scoredfor clinical signs of disease 2-3 times per week. The arthritic scorescale is defined in Table 7.

TABLE 7 Severity Score Gross Pathology 0 No evidence of erythema andswelling 1 Erythema and mild swelling confined to the mid-foot (tarsals)or ankle joint 2 Erythema and mild swelling extending from the ankle tothe mid foot 3 Erythema and moderate swelling extending from the ankleto the metatarsal joints 4 Erythema and severe swelling encompass theankle, foot, and digits Each paw is assessed individually and thearthritic score for a given mouse is the sum of the scores for all paws(maximum score of 16)

Treatment with ATG alone resulted in a reduction in disease scores andthe addition of latent TGF-β1 did not appear to provide an additionalbenefit under the conditions tested. The results are shown in Table 8.

TABLE 8 Arthritic Score (Mean ± SEM) Rabbit Ig + ATG + Day Rabbit IgLatent TGF-β1 ATG Latent TGF-β1 22  0.0 ± 0.0  0.0 ± 0.0 0.0 ± 0.0 0.0 ±0.0 26  2.7 ± 2.3  3.7 ± 2.5 2.8 ± 2.6 5.0 ± 3.5 29 10.0 ± 4.2 10.1 ±4.3 3.4 ± 4.9 5.8 ± 5.3 32 10.5 ± 4.4 11.6 ± 3.0 5.5 ± 6.0 6.6 ± 5.1 3510.5 ± 4.4 12.0 ± 2.7 6.5 ± 5.9 7.6 ± 4.3 39 10.9 ± 3.9 12.9 ± 2.0 7.0 ±5.5 8.1 ± 3.3 41 11.0 ± 4.1 12.5 ± 2.8 7.3 ± 5.2 8.8 ± 3.6 43 11.7 ± 3.713.4 ± 1.8 7.5 ± 5.4 8.9 ± 3.8 46 11.5 ± 4.5 13.4 ± 2.8 9.5 ± 5.1 9.1 ±3.5 50 12.7 ± 3.0 13.5 ± 2.1 9.5 ± 4.9 9.0 ± 3.7 53 12.4 ± 4.0 13.8 ±2.1 9.6 ± 4.8 9.3 ± 3.7 57 12.4 ± 4.0 13.8 ± 2.1 9.6 ± 4.8 9.3 ± 3.7

The collagen-induced arthritis is a short-term animal model, in whichthe treatment takes place on a timescale of weeks, versus months for thelupus model. This shorter timescale might be insufficient to observe thebenefit added by administering TGF-β1 with the ATG, which was seen inthe lupus model. Thus, different dosing regimens or further testing ofadditional animal models may show benefits of combined administration ofATG and latent TGF-6.

Murine Model of Uveitis

The effect of ATG+/−TGF-β1 was tested in a mouse model of uveitis. Toinduce disease, B10.RIII mice (Jackson Laboratory) were immunizedsubcutaneously on day 0 with 100 μg of amino acids 161-180 of humaninterphotoreceptor retinoid binding protein (IRBP₁₆₁₋₁₈₀) (customsynthesis, New England Peptide) in complete Freund's adjuvant at twosites (between shoulder blades and in pelvic region). Starting on day10, funduscopic examinations were performed on individual mice and adisease score was assigned. To perform the examination, the eyes of micewere dilated using one or two drops of Mydriacyl™ 1% (Cat. No. 1120, JAWebster) and rested in a darkened room for approximately five minutes.Mice were manually restrained and the retinas of both eyes visualizedusing an indirect ophthalmoscope with a 78 diopter lens. The eyes werescored for inflammation using a progressive scoring system between 0 and5, as described in Table 9.

TABLE 9 Score Gross Pathology 0 Normal retina 1 Vascular inflammationproximal to the optic nerve 2 >10 inflammatory lesions confined to onequadrant of the eye 3 >10 inflammatory lesions in more than one quadrantof the eye 4 Inflammatory lesions are contiguous 5 Retinal detachment

Treatment with ATG or control rabbit IgG was initiated at disease onset(score of 1) and consisted of two i.p. injections of 500 μg (˜25 mg/kg)delivered four days apart (days 10 and 14). Latent TGF-β1 was given fromdays 15-27 as thirteen daily i.p. injections of 4 μg per mouse. A 4 μgdose of latent TGF-β1 corresponds to a 1 μg (˜0.05 mg/kg) dose of theactive (mature, non-LAP-associated) portion of the molecule. Thetreatment groups included (1) phosphate buffered saline (PBS) control(2) control rabbit IgG, (3) control rabbit IgG+latent TGF-β1, (4) ATG,and (5) ATG+latent TGF-β1, with six animals per group. Treatment withATG alone resulted in a reduction in disease scores and the addition oflatent TGF-β1 did not appear to provide an additional benefit under theconditions tested. The results are shown in Table 10.

TABLE 10 Uveitis Score (Mean ± SEM) Rabbit Ig + ATG + Day PBS Rabbit IgLatent TGF-β1 ATG Latent TGF-β1 8 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.000.00 ± 0.00 0.00 ± 0.00 10 1.25 ± 0.16 1.22 ± 0.15 1.29 ± 0.18 1.13 ±0.13 1.13 ± 0.23 14 4.00 ± 0.00 3.17 ± 0.32 3.67 ± 0.22 2.10 ± 0.48 2.17± 0.34 17 3.81 ± 0.10 3.00 ± 0.35 3.58 ± 0.29 1.75 ± 0.37 2.00 ± 0.37 213.81 ± 0.10 2.58 ± 0.34 3.33 ± 0.33 1.92 ± 0.42 2.08 ± 0.31 24 3.94 ±0.06 2.58 ± 0.42 3.50 ± 0.34 2.00 ± 0.41 1.92 ± 0.36 29 3.75 ± 0.11 2.27± 0.47 3.33 ± 0.33 2.08 ± 0.42 2.00 ± 0.40 35 3.88 ± 0.09 2.25 ± 0.463.33 ± 0.40 1.92 ± 0.40 2.00 ± 0.35 46 3.56 ± 0.13 2.09 ± 0.41 3.25 ±0.39 2.00 ± 0.44 1.92 ± 0.40 56 3.69 ± 0.12 2.17 ± 0.44 3.25 ± 0.39 1.67± 0.38 1.75 ± 0.25

The uveitis model is a short-term animal model, in which the treatmenttakes place on a timescale of weeks, versus months for the lupus model.This shorter timescale might be insufficient to observe the benefitadded by administering TGF-β1 with the ATG, which was seen in the lupusmodel. Thus, different dosing regimens or further testing of additionalanimal models may show benefits of combined administration of ATG andlatent TGF-β.

All publications, patents, patent applications, and biological sequencescited in this disclosure are incorporated by reference in theirentirety.

1. A method of treating a mammal with an autoimmune disease, the methodcomprising: (a) depleting circulating lymphocytes in the mammal, (b)allowing the lymphocytes to begin repopulating, and (c) during therepopulation phase of (b), administering to the mammal a therapeuticallyeffective amount of latent TGF-β and/or another agent that promotes theexpansion of regulatory T cells.
 2. The method of claim 1, wherein themammal is a human.
 3. The method of claim 2, wherein the autoimmunedisease is multiple sclerosis.
 4. The method of claim 1, wherein thelymphocytes depleted are predominantly T cells.
 5. The method of claim1, wherein the lymphocytes are depleted by administering an agent chosenfrom the group consisting of anti-thymocyte antibody, anti-CD52antibody, and anti-CD3 antibody.
 6. The method of claim 5, wherein theanti-thymocyte antibody is chosen from the group consisting of rabbitanti-thymocyte globulin and equine anti-thymocyte globulin.
 7. Themethod of claim 1, wherein the regulatory T cells are CD4⁺ CD25⁺ Tcells.
 8. The method of claim 1, wherein latent TGF-β comprises matureTGF-β and one or both of the following: (a) latency associated peptide(LAP); and (b) latent TGF-β binding protein (LTBP).
 9. The method ofclaim 1, wherein latent TGF-β is latent TGF-β1.
 10. The method of claim1, wherein latent TGF-β is administered systemically.
 11. The method ofclaim 1, wherein the agent that promotes expansion of regulatory T cellsis one or more agents chosen from the group consisting of: (1) IL-10,(2) IL-4, (3) IFN-α, (4) vitamin D3, (5) dexamethasone, and (6)mycophenolate mofetil.
 12. The method of claim 1, wherein the agent thatpromotes expansion of regulatory T cells is rapamycin.
 13. The method ofclaim 1, wherein the autoimmune disease is associated with a loss ofkidney function and the treatment results in slowing of the loss of orimprovement in kidney function of the mammal.
 14. The method of claim13, wherein the slowing of loss or improvement in kidney function isindicated by a change in systemic blood pressure, proteinuria,albuminuria, glomerular filtration rate, and/or renal blood flow. 15.The method of claim 13, wherein the autoimmune disease is Goodpasture'ssyndrome, Wegener's syndrome, IgA nephropathy, IgM nephropathy, oranother autoimmune disease that impairs kidney function.
 16. A method oftreating a mammal with an autoimmune disease comprising: (a)administering a lymphocyte-depleting antibody to the mammal, therebyreducing the population of peripheral blood T cells; and (b)administering latent TGF-β to the mammal in an amount effective to slowthe progression of the disease and/or improve symptoms.
 17. The methodof claim 16, wherein the mammal is a human.
 18. The method of claim 16,wherein the autoimmune disease is multiple sclerosis.
 19. The method ofclaim 16, wherein the lymphocyte-depleting antibody is anti-thymocyteantibody, anti-CD52 antibody, or anti-CD3 antibody.
 20. The method ofclaim 16, wherein latent TGF-β is latent TGF-β1.